sheep showed primed superoxide anion release compared with controls when ...... Jacob,. H.S.. (1990). Endothelial cell platelet-activating factor primes neu-.
In vivo endotoxin without
maintenance
short-term
A. Carey,
Department Medical
to
of
of sheep
Kurt
H. Albertine,
Physiology
College,
increases
of primed
cultures
Lisa
Abstract: lished responses endotoxin
exposure
and
Thomas
Division
Jefferson
of
superoxide
bone
and
Marlys
Pulmonary
University,
neutrophil anion
release
H. Gee
Medicine
and
Critical
Care,
Department
of
superoxide
respond
compared
with
controls
when
stimulated with phorbol myristate acetate. Priming was no longer present in neutrophils tested after 2 days in cubture. The neutrophil cell count, however, was higher after 2 days in culture than in controls. Increased neutrophil production/viability was reproduced in naive bone marrow cultures by replacing part of the normal serum medium with plasma taken from sheep several hours after endotoxin infusion. These data suggest that a dissociation exists between tion/viability
priming and increased in response to endotoxin.
56:
1994.
145-150;
Key bility
neutrophil
J.
endotoxin . sheep #{149} bone marrow . superoxide anion release
Words: . priming
. neutrophil
.
to activating
MATERIALS Culture
of of
medium was prepared as described by Greenberg et al. Briefly, the following were added to 800 ml of McCoy’s 5A media: 6 ml of7.5% NaHCO3, 20 ml ofsodium pyruvate (100 mM), 4 ml (lOOx) of minimal essential medium (MEM) vitamin mixture, 8 ml of Eagle’s MEM amino acid, 4 ml (lOOx) of Eagle’s MEM nonessential ml of 21 mg/ml L-serine, 1.6 ml of 10 mg/ml 5 ml of glutamine, 250 mg of streptomycin, penicillin, 142 ml ofsterile fetal bovine serum ml of sterile horse serum (HS) to achieve a 12.5% HS medium. All chemicals were
via-
Gibco endotoxin
physiological
in
experimental
responses,
functional response of inflammatory phages and neutrophils. We and that peripheral blood neutrophils
animals including
changes
causes in
BRL,
further
characterizing
the
cells such as macroothers have demonstrated isolated hours after en-
duration
of
this
The
Collection Bone sterile
Island,
NY.
The
medium
was
filter-
use.
relative maturity of the cells, neutrophils, that are capable of which cause priming is unknown. of the duration of priming in bone short-term culture method for sheep purpose was to determine the dura-
of
1 ml 9 ml
was drawn in anesthetized
heparin
t\t)t)rt’viat
was
injected
inns:
C : P. (-Utst
st-ruin;
v1EN1, Rrpn Collegt-,
IN
acetate; nt
request
Ihis
New Received
IN
crest a lung
needle,
and
during lymph
the
Jeflerson
Health work
F, t ii taut Hall
current
Sciences was
Rooni
431.
address:
Center, in part
presented
I)t’pt
. (‘t’.
I-IS,
actor:
optical
FBS. Ic(N4-CSF,
actor; (I(’nsitv;
horse
seroiris;
PN’IA_
photbol
factor.
necrosis
H
Alutnni
ing
()l),
other
for the contents
1)liStthi
I ). PSt(’t5(ItXit5
colonv-stitnulattng
tiiediutn;
s: NI arlvs
19107. H. Albertine’s
Utah
l)l1sttsa:
granulocvte
(‘ss(’ntial
Kurt
Journal
the
(olonv-stiiosulat
tninitnal
ti)VtIstate
in
the right iliac sheep to prepare
through
ml
S1-I
(;-(
granulocyt(’-rnacrophag(’
of
from
was retained in the syringe as an anticoagulant of bone marrow drawn into the syringe. The
toil bovine
PA
of bone marrow
and place vascular lines [5]. The bone marrow was into a 13-gauge Jamshidi needle with an adaptor to 10-mb syringes were attached. Each of the six to eightused contained 2 ml of sterile heparin. The first 1
syringes ml
and culture
marrow surgery
fistula drawn which
neutrophil
functional change. Neutrophils are short-lived cells with a circulating half-life of6 to 8 h [3]; therefore, it is evident that blood samples taken 24 h after endotoxin infusion contain leukocytes that were released from bone marrow during or after the endotoxemia. from stem cells to mature responding to the signals To address the question marrow, we developed a bone marrow cells. Our
Grand
before
amino acids, 0.4 L-asparagine, 250,000 units of (FBS), and 142 12.5% FBS and purchased from
a the
dotoxin infusion show primed superoxide anion release when stimulated in vitro [1, 2]. Priming is also evident in sheep bone marrow neutrophils isolated 24 h after a bolus endotoxin infusion [1]. Sustained priming of superoxide anion release, particularly in immature bone marrow cells, suggests that this functional modification of neutrophils may be part of a long-term inflammatory response to endotoxin. Given the persistence of priming in both peripheral blood and bone marrow-derived neutrophils, we became interested in
AND METHODS
media
sterilized Infusion
to
[4].
[4].
INTRODUCTION
myriad
agents
access to large numbers that are physiologically content and ability
The
producBiol.
Leukoc.
Jefferson
tion of priming by culturing bone marrow from sheep after exposure to endotoxin. We modeled our system after that described by Greenberg et al. [4], which is a long-term liquid culture method. This method is particularly well suited for
our studies, because it allows ready of bone marrow-derived neutrophils normal in terms of their granular
release
Medicine,
Pennsylvania
from surgical control animals and from sheep given a 12-h infusion of endotoxin. In these bone marrow aspirates, neutrophils from endotoxin-treated sheep showed primed
anion
in
marrow
Philadelphia,
A short-term bone marrow culture was estabdetermine the functional and proliferative of neutrophils sampled from sheep exposed to in vivo. Iliac crest bone marrow was drawn
counts
.
of Physiology, 1020
locust
I)epartment
Salt
lake
at the
.Jr’tIrson Street,
of Pediatrics,
City,
Utah,
Experimental
Medical
Philadelphia, University
84132-1001. Biology
93
meeting
Orleans. Novetsiber
of Leukocyte
16,
1993;
Biology
accepir(1
Volume
Ntarch
56,
31.
August
199-1.
1994
145
of the
syringes
centrifuge calcium-free centrifuged
were
transferred
tubes each balanced for 15 mm
to two
sterile,
containing 5 ml salt solution (Gibco). at 900g in a Beckman
capped
of
50-mb
sterile Hanks’ The tubes were model TJ-8 cen-
trifuge.
The buffy coat was aspirated, subjected to hypotonic to remove the red blood cells, and then resuspended sterile, 2 x Hanks’ balanced salt solution (Gibco) to the
nucleated
cells
to isotonicity.
The
cells
were
lysis in return
centrifuged
at 2500g in a Sorvall refrigerated centrifuge. The washed twice with McCoy’s modified 5A medium trifuged again at 2500g. The cells were resuspended of medium and the nucleated cells were counted
pellet was and cenin 10 ml using a
Peripheral
blood cultures
To determine under these blood-derived
the viability of peripheral blood culture conditions, we cultured white blood cells. Forty-five
sheep blood was obtained
was drawn as described
McCoy’s row cells.
5A medium The cultures
counted,
and
marrow
5 ml above,
counts
exposure
drawn
from
sheep during sterile surgery periments. These animals
of
of heparin. The buffy coat and the cells were plated in
as previously described were harvested at
differential
In vivo endotoxin Bone
in
neutrophils peripheral milliliters
for bone 2 days, the
marcells
obtained.
protocol the
right
iliac
crest
of
normal
served as controls for these exwere allowed to recover for 4 to 5
hemacytometer. Some nucleated red blood cells survived hypotonic lysis and were present in the culture. The cells were seeded in a 20-mb volume in a T-75 cell culture flask (Corning Plastics, New York, NY) at a concentration of 4000 cebls/il. A portion of the cells was saved for a baseline differential cell count using Wright-Giemsa-stained slides
days before the endotoxin consisted of a low-dose,
and a superoxide anion remainder of the cells tamed at 33#{176}Cin room 2 days.
prepared for a sterile necropsy. At this time, bone marrow was drawn from the left iliac crest and cultured as described above. When a bone marrow aspirate was not successfully obtained at the time of surgery, a bone marrow aspirate was
Superoxide
anion
release assay as described below. The were placed in an incubator mainair supplemented with 3% CO2 for
release
assay
Bone marrow cells, freshly aspirated or cultured, were trifuged at 2SOOg and resuspended in sV-2 ethanesulfonic acid (HEPES) buffer containing (in mM): 140 NaCl, HEPES, 10 KC1, 0.1 CaCl2, 0.2 MgCl2, 11.0 NaHCO3, glucose, and 14 iM albumin (Sigma, St. Louis, MO), 7.4. Because the bone marrow contained heterogeneous types, we diluted the cells to a concentration of 250,000 what we termed superoxide anion-releasing cells
cen20 5.0 pH cell of (eo-
sinophils, bands, and segmented neutrophils/50 jl) and plated them in Nunc high-binding microtiter wells. The cells were allowed to settle for 10 mm. The wells were stimulated with 50-l test solutions containing 10” through 10 M phorbol myristate acetate (PMA; Sigma) and 0.32 mM ferricytochrome c (type III from horse heart, Sigma). Triplicate wells received each treatment. Three wells received ferricytochrome c and HEPES buffer to measure basal release of superoxide anion. There were also three assay blank wells that contained ferricytochrome c, HEPES buffer, and 700 U superoxide dismutase (bovine erythrocytes, Calbiochem) to confirm that by superoxide
the
ferricytochrome dismutase.
c reduction
was
inhibitable
Changes in optical density (OD) were measured intermittently on a microplate reader (Dynatech MR 600) using a test wavelength of 550 nm and a reference wavelength of 670 nm. Superoxide anion release was calculated using the conversion proposed by Pick and Mizeb [6]: nmol O2 = [(test OD
-
reference
Collection After bator. medium
OD)
x
100]
6.3.
at 2 days
2 days,
the
culture
The adherent using a cell
flasks cells scraper
were
removed
from
were gently scraped (Gibco). A cell count
the into was
incuthe ob-
tamed and percent viability was determined using trypan blue exclusion. Cell counts were reported for viable cells only. Smears were made for total cell differential counts. The cells were centrifuged at 2500g for 5 mm, and the cell pellet was resuspended in HEPES. The cells were plated in microtiter wells as described above and a superoxide anion release assay was completed.
146
Journal
of Leukocyte
Biology
Volume
56,
August
1994
infusion was 12-h infusion
(Sigma) at 10 ng/kg-min, as described After a 12-h recovery period, the sheep
begun. of E.
The infusion coli endotoxin
by Sloane et was anesthetized
al.
[7]. and
drawn from a control animal. Because the sheep used for our studies are raised domestically, they are subject to environmental stresses that change seasonally. Bone marrow characteristics may also vary with the age of the animal. To avoid issues of age and seasonal variation, we chose age-matched and seasonally matched sheep to serve as controls when a bone marrow aspirate was not successfully obtained at the time of surgery.
Replacement
studies
To determine whether the effects of in vivo endotoxin infusion on these bone marrow cultures are attributable to a plasma-borne factor, we collected heparinized blood from two sheep 7 h after the end of the 12-h endotoxin infusion, at a time point characterized by peripheral blood leukocytosis [7]. The blood was centrifuged for 15 mm at 900g in a Beckman model TJ-8 centrifuge to separate the blood components. Plasma was then removed and frozen. The plasma samples from both sheep were pooled, combined with an equal volume of FBS, filter sterilized, and frozen in 5-mi aliquots until needed. Control plasma was collected from two sheep, after surgery but before experimental intervention, and was likewise processed and combined with an equal volume of FBS. Bone marrow was then drawn from a surgical control animal and subjected to the normal separative procedures. The nucleated cells were suspended in serumbess medium and were plated in three T-75 flasks containing the following combinations with 12.5% FBS: 12.5% horse serum (HS), the normal culture medium; 12.5% postendotoxin plasma (END); and 12.5% control plasma (CP). An initial cell superoxide anion release assay was obtained, as were superoxide anion assays on the contents of the three flasks after 2 days in culture. Total cell count, differential, and percent viability
were
also
obtained
In vitro endotoxin
as
described
above.
studies
To determine whether incubation with endotoxin in vitro would change neutrophil cell function and viability, we added E. coli endotoxin in I ml of saline to naive cultures to achieve final endotoxin concentrations of 10 ng/250,000 nucleated cells (160 ng/ml) and 0.1 ng/250,000 nucleated
cells (1.6 ng/ml). to be associated while the lower plate
The higher concentration has been shown with neutrophil respiratory burst activation, dose is associated with priming [8]. A third
received
differential, tamed for
saline the
to serve
as a vehicle
and superoxide three treatment
anion groups
control.
release after
Cell
assays 2 days
TABLE
Properties
of
were obin culture.
Initial studies were conducted to characterize the culture systern. We were able to support granulocyte production for 14 days using a biweekly medium replacement schedule. This suggests that the culture conditions were compatible with stem cell survival and colony formation. Table 1 summarizes the data gathered from surgical control bone marrow cubtures harvested after 2 days. Note that neutrophil counts This where
is in conthe neu-
trophil counts dropped 71% ± 8% over the 2-day period. The ability to release superoxide anion in response to PMA was maintained over the 2 days in culture, suggesting that the neutrophils which were present at 2 days were healthy and were not down-regulated or primed by the culture conditions. PMA-stimulated superoxide anion release assays were performed on unfractionated bone marrow samples that were not subjected to any separative procedures to isolate the neutrophibs. marrow
We samples
performed some using discontinuous
separations of Percoll/plasma
the
bone gra-
dients as previously described [9] before determining the superoxide release. Release from these isolated granubocytes was significantly higher than the release from unfractionated bone marrow: 4.05 ± 0.6 nmol O2 versus 2.25 ± 0.3 nmol O2 (n = 10, P < .05, t-test, two-tailed). An abbreviated version ofour superoxide anion assay requires 3-4 million neutrophils. Given that these cultures normally yield approximately i8 million neutrophils after 2 days, we were not always able to obtain enough cells in any one layer of the gradient to perform the superoxide anion assay. For this reason, we used unfractionated bone marrow in the assays, even though we found higher release from fractionated cells.
-0---
-
Marrow
Short-Term
Cultures
Value
Fresh
marrow’
2-day
bone
marrow
4000 39
(P
unpaired
Bone
3
±
133 0.34
±
from 1-test,
marrow
1487
±
127
61
±
2’
885 2.27
±
80’
±
0.35
granulocytes)
difference
.05,